Bone is a naturally regenerative tissue possessing the capacity of healing from injury by recapitulating the embryonic skeletal developmental process. However, an estimated 5-10% of fractures fail to recover properly and proceed to delayed union or nonunion due to mechanical instability and/or poor biology (1). The mechanical problem is the one relatively easy one to be solved by external or internal fixation, but the biological issue remains a significant challenge (1). There are few fractures had tendency to undergo process of nonunion due to their unique anatomy. These include femur neck, scaphoid, talus neck, and Jone's fractures.
Femur neck fracture is one of the most common fractures treated surgically in orthopedic surgery. It comprises more than 50% of the hip fractures which is estimated to exceed 500,000 in United States by year 2041 as the population aging (2-34). Surgical fixation of the femur neck fracture is extremely challenging especially when fracture presents in elderly and fracture displaced. Due to poor bone quality and healing power in the elderly, higher rate of fixation failure including mal-union, nonunion and AVN had been reported in the literature in this group which causes high re-operating rate and increasing of the total cost (5, 6). Thus, hemiarthroplasty has been advocated by some surgeon (7). Despite the increasing cost for internal fixation, interestingly, more patient choose internal fixation over hemiarthroplasty based on recent cost-benefit analysis using the willingness to pay technique (8).
Mainstay of current internal fixation of femur neck fracture consists of lag screws and DHS. Lag screw fixation is the most popular technique used for femur fracture in United States. Most surgeons use 3 cancellous screws in an inverted triangular orientation. The inferior screw is recommended to be placed close to the calcar and anterior and posterior superior screw should put close or within 3 mm of the cortex to ensure better purchase. The cannulated screws are used with assistance of guide wire for parallel placement which allows dynamic compression. Lag screw fixation has advantage of minimally invasive insertion, limited disruption of femur head blood supply and superior torsional stability (9). However, strength of lag screw fixation is dependent on the quality of bone on the lateral wall of the proximal femur. Severe osteoporosis in elderly compromise strength of bone in support the screw fixation or in the fracture with vertical fracture line which imposes larger deforming force on the screw surpassing the capacity of screw fixation leads to fixation failure (7).
The challenge of fixation of femur neck fracture is not only come from the poor quality of the bone in the elderly but also from the biology of bone healing. Bone healing is slower in elder patient per se due to senesces of stem cells. The situation worsen in femur neck fracture because the femur neck is enveloped by synovial fluid, thus no periosteal blood supply and fracture disrupting the intraosseous blood circulation to the proximal fragment. Thus, any means to facilitate re-vascularization and bone healing will help to win the battle for racing between loss of reduction and bone healing.
Scaphoid is divided into proximal and distal poles, a tubercle, and the waist. eighty percent of the scaphoid is covered with articular cartilage. The main vascular supply is derived from scaphoid branch of the radial artery entering the dorsal ridge and supply 70%-80% of the scaphoid, including the proximal pole. Scaphoid fracture is the most common carpal fracture. Fracture through the waist or proximal pole disrupted the blood circulation, thus like femur neck fracture, had high incident of delayed or nonunion. Thus, screw fixation is recommended to achieve stable fixation thus revascularization of the proximal pole. Recently, percutaneous technique is advocated to preserve the circulation during the surgery (Kenneth, J Koval ed Hand book of fractures).
Talus fracture is the second in frequency among all tarsal fractures. Similar to the femur neck and scaphoid, majority (70%) of the talus is covered by articular cartilage No muscles originate or insert into the talus. The vascular supply to the talus depend on facial structures to reach talus, thus, in Hawkins' 3-4 talus neck fracture, capsule is disrupted which result in osteonecrosis (Keneth Koval ed Hand book of fractures).
The metadiaphyseal region of the 5th metatarsal is an area of circulatory watershed resulting in limited blood supply. Fracture that occur at the meta-diaphyseal junction about 1.5 to 2.5 cm distal to metatarsal base, are commonly called Jones fracture. Because the comprised blood supply in this area, the fracture is at risk of nonunion. Thus, screw fixation is recommended for athletes to minimize the possibility of nonunion and prolonged restriction from activity (Jay Lieberman AAOS comprehensive review).
Tendon and ligament injury is the common injury in the sport. Healing of the ligament and tendon to the bone tunneling after reconstruction is the slow process resulting in prolonged restriction activity for the athletes to return the games. Speeding up the healing would be significantly beneficial to the survivorship of the graft and the athlete career.
Burst fracture is the fracture compression and flexion injury to the spine lead to the crushed of the vertebrate. The fracture are involved both anterior and middle column of the spine and is considered unstable fracture. The treatment includes the posterior pedicle screw fixation and possible anterior spine fusion. Pedicle screw can efficiently reduce the fracture of the vertebrate body by ligament taxis in most of time. The anterior spine fusion is only necessary to increase strength and support to anterior middle column to release the strain of the pedicle screw so that the later would not fail before the fracture healed. Thus, any means to facilitate the fracture healing may eliminate the need of anterior fixation.
Taking together, the unique injuries described above call for a biological active fixation device that can provide the mechanical strength and had capacity to stimulation healing.
Bone morphogetic proteins (BMPs) are a class of the proteins belonging to TGF beta superfamily and exhibiting potent osteoinductive activity in vivo. BMPs initially identified by their ability to induce ectopic, endochondral bone formation (Urist, 1965). Since then, 18 members of the BMP family have been identified. Structure and characterization of these proteins had been reviewed in detailed in recent patent application. US patent No 20090169592 “Osteogeneic devices and method of use thereof for repair of endonchondral bone and osteochondral defect”. BMPs can be isolated in relative pure form by processes described in U.S. Pat. No. 4,294,935, 1981 and U.S. Pat. No. 4,455,256, 1983. Recently, Recombinant human BMP2 and BMP7 is made available by technique described in U.S. Pat. No. 6,150,328, 2000 and U.S. Pat. No. 7,459,527, 2008. Both BMP2 and BMP7 are potent osteoinductive agents, BMP2 either alone or in combination of other graft materials had shown to heal some critical size bone defect or nonunions. BMP2 and BMP7 are currently commercially available and FDA approved for treatment of the lumbar spine fusion and nonunion of the tibia fractures. BMP12, unlike BMP 2 and BMP7, exhibit the potent potential for tendon and ligament regeneration. Although BMPs is able to induce osteogenesis, or tendon/ligament genesis, very high dose is required to achieve such effect if use alone. To efficiently delivery of BMPs and improve their bioavailability, finding a suitable carrier is the major focus of the current research.
Search for the appropriate carrier for delivery of the BMPs started as early as discovery of the methods to purify MMPs. U.S. Pat. No. 4,526,909 to Urist, 1985 disclosed to used polymethylmethacrylate as delivery system for BMPs however, this system proved not working. BMPs are hydrophilic whereas, PMMA is hydrophobic. Most of the BMP will be trapped inside of the PMMA except the outer surface which can release by a leaching mechanism. Furthermore, monomer of PMMA and heat generated by polymerization will denature the protein thus inactivate the osteoinductive active of the BMPs. U.S. Pat. No. 4,596,574 to Urist, 1986 disclosed to use a biodegradable porous ceramics including tricalcium phosphate as carrier to deliver BMPs, it turn out to be great idea, porous ceramic not only able to control drug release but also attract stem cell in-growth thus augment host tissue integration. Porous ceramic is still use in combination of the organic polymers for drug delivery (U.S. Pat. No. 6,949,251). U.S. Pat. No. 4,563,489 disclosed of methods using biodegradable organic polymer, polylactic acid polymer as carrier for delivery of BMPs. it was proposed to use biodegradable material to make implant such screws. The idea is attractive because feature of biodegradability eliminate the possibility of hardware removal, however, this is not feasible because toxic degradation product and lack of mechanical strength. Since the early effort, many new materials have been developed as carrier for BMP delivery. The materials can be classified into two categories organic or inorganic. Inorganic carrier further divided as biodegradable or un-degradable. Most of organic carriers are biodegradable which further divided into hydrophobic or hydrophilic. These materials are used alone or in combination to control the rate BMP release so that bone formation is balance with rate of the biodegradation of the implant without de mechanical strength. The use of these materials is fully elucidated in following US patents (U.S. Pat. Nos. 6,110,484, 6,949,251, 6,544,290, 7,049,348, 7,192,604, 6,949,590). Of all materials tested, however, gelatin is still most promising carrier.
Gelatin is nature polymers derived from denature of type I collagen. Gelatin derived from bovine or porcine has been widely used in pharmaceutical industry as capsule materials and stabilizer, gelatin also used as implant such gel-foam to stop surgical bleeding and demonstrated non-toxic, excellent biocompatibility and biodegradability. More importantly, Both BMP2 and BMP7 bind to the gelatin derived from bone, thus, better retain BMPs in the carrier. Porous form of gelatin is easy accessed by water and stem cells thus serve better as a carrier for BMPs. Recently, FDA approved clinical application of the porous collagen film as a carrier for BMP to enhance spine fusion (BMP-2) or healing of bone defect in nonunion (Medscape today, Carriers for BMPs). US patent NO. 20080294085 to Stamps disclosed collagen sponge rolled biphased ceramic to treat the precollapsed oateonecrosis of the femur head.
With advanced of nanotechnology, gelatin has been used to form a nanoparticles, natotubes and nanofibers. Natoparticle refers to the particles with diameters ranging from 10 nm to 1000 nm. Using nanoparticles as drug delivery system confirms sustained drug delivery, and subcellular action, therefore, significantly enhances the bioavailabilty of the drug. Preparation of gelatin nanoparticles is very simple by two step dissolvation methods. Gelatin nanoparticles are also easy to be crosslinked and chemically modified. Therefore, it has immense potential to be used for as colloidal drug delivery system. Other advantages are: It is inexpensive and can be sterilized (Mohsen, 2008). Study of BMP2 gelatin nanoparticles demonstrated that gelatin nanoparticles stabilize BMP2 for sustained release for a prolonged period of time (6 weeks in vitro and in vivo (Kempen et al Biomaterial 2008). Recently, human recombinant gelatin (rHG) is available as a source for preparation of nanoparticles to decrease risk of mad cow disease and lower the antigenicity. hHG nanoparticles exhibited good biocompatibility and controlled release of FITC-BSA (Young, 2009). Gelatin nanoparticle also exhibits protective effect of basic FGF (Tsutomu, 2007). In present invention, we use gelatin nanoparticle as a carrier for MMPs.
Stem cells refer to the cell able to differentiate into variety of lineage, yet maintain the ability to self renew. Mesenchymal stem cells (MSCs) are the pluripotent or multipotent stem cells capable of differentiating into osteogenic, chondrogenic, adipogenic and other mesenchymal lineages in vitro. MSCs are no specific marker, thus laboratory defined MSCs remains debatable. It is consensus that MSCs does express CD90 and CD105 but not express CD34 and CD 45. This may help for isolation of the MSCs. MSCs existed in many tissues including, adipose, muscles, liver, blood, as well as barrows. Bone marrow is the one of the richest resource for stem cells, and the most frequent donor site for harvesting stem cells for clinical use. The stem cells isolated from bone marrow refer to bone marrow stem cells (BMSCs). BMSCs are osteogenic, implantation of human BMSCs with a carrier into a critical size of bone defect which usually undergoes nonunion healed completely with short amount of the time (U.S. Pat. No. 686,900 to Kadiyala, 2005). Both autologous and allogenic MSCs can be used for orthopaedic surgery, autologous cells are preferred because it is non-immunogenic, easily accessible, sufficient resources available (only need 60 ml v.s 1.5 liter for other purpose). Standard procedure for isolation and purification of the MSCs from bone marrow consists of several steps: collection of the bone marrow aspirates, separation of nucleated cells from red cell, and purification of MSCs from other nucleated cells, clone selection and characterization as it delineated in the U.S. Pat. No. 7,015,037 to Furcht at el, 2006. Purification of the MSCs stem cells takes a few weeks to months to complete which renders multiple procedure and inconvenience for clinical use. Multiple purification steps in an open system can raise the risk of infection. In addition, in vitro differentiation and mutation is also a concern. Thus, application purified MSCs appears less attractive (Marcus et al 2009).
Collection and application of the bone marrow aspirates for bone regeneration and repair is a well know arts for centuries. However, clinical outcome is not consistent. Recent studies have shown that the key to effective clinical outcomes using MSC therapy is to provide that sufficient number of the MSCs cells to the patient which repairs the bone or tissue defects. This refers to “Regenerative MSC Threshod” (Kadiyala, 2005). Bone marrow aspirates are usually collected from the iliac crest. It contains red blood cells, lymphocytes, granucytes, monocytes, fibroblasts, stem cells including bone stem cells which possess only very small percentage of all cell populations. Therefore in order to achieve Regenerative MSC Threshod, a method needs to enrich MSCs in the bone marrows aspirates, a process often referred as concentration. Ficoll gradient centrifugation is well known art for making bone marrow aspirate concentrates (BMAC). However, this is an open system required special equipment, and time consuming (need at least 45 to 1 hours), required appropriate trained personnel. U.S. Pat. No. 6,981,948 to Pellegrino 2006 disclosed a bone marrow aspiration system in which bone marrow aspirates allowed to pass through chamber containing graft substrates during the collection procedure. The cell retains in the graft material and ready for implantation. The attraction of this design is simple, quick, efficiency, and in the closed system. However, this system fails to separate the richest red blood cells from other cells. U.S. Pat. No. 424,973 to Augustus disclosed a 17.3% glucose gradient centrifuge for concentration of bone marrow aspirates which only need 10 minutes, however, like other gradient centrifuge methods, it is an open system and require special equipment. Recently, a new closed Harvest BMAC system can complete concentration in 10 to 15 minute and available in USA. Recently, Asian described an immuno-isolation to purify MSCs without culture results in 60 times increase in fibroblast CFU as compare those without purification. One aim of the present invention is to provide a clinical usable approach to further enrich MSCs.
In addition to the number of the MSCs, like BMPs, methods of delivery and retention of the MSCs in the site of action is also important for clinical application of MSCs osteogenesis. Many carriers have been tried for delivery of MSCs but most of them are lack of ability to retain MSCs. Thus majority of stem cell migrate away through the blood circulation after implantation decrease its bioavailability. Since MSCs is alive, It significantly limited the materials which can be used to encapsulate the cell in their matrices.
Carrier for stem cells is limited with respect of cell retention, most of the carriers are pre-casted, thus retention of the cell is a problem unless the carrier modified with special affinity molecule. e.g. RGD. In situ polymerized implant has advantage of cell retention, however, heat generation during polymerization and toxic monomers create hostile environment for live cell to survive. Thus, few polymers qualified as in situ polymerizing system for cell delivery (U.S. Pat. No. 6,110,484 to Sierra). Chitosan and fibril sealant are two of few candidates.
Chitosan is a biocompatible nature polymer developed as a delivery vehicle for the peptide (U.S. Pat. No. 2008213354 to Sung). It exhibits thermosensitive property in presence certain inorganic phosphate salts such as glycerol phosphate or tiacalcium phospate, the liquid chitosan solution transformed into a gel at clinically feasible time ranging from 2 to 10 minutes at 37° C. Chitosan have been used extensively for chondral cell transplantation (Marchand, 2009) Chitosan will serve as an alternative for delivery of MSCs In present invention.
Fibrin sealant as delivery system for drugs including growth factors and stem cells is a well known art in the field. Variety applications of fibrin sealant have been nearly exhausted in a world patent (WO/1996/040174, Supplement and un-supplemented tissue sealants, methods of their production and use). Fribrin sealants are two component tissue adhesive systems mimicking natural clot cascade. Two components of fibrin adhesives are stored in sterilized lyophilized powder. The components are reconstituted into liquid form by adding distilled water. As two component mixed together, they polymerized at the site of application into a relatively dense gel. Thrombin in combination of Ca2+ catalyze polymeration of fibrinogen, converting the fibrinogen into fibril polymer. Further, thrombin and Ca2+ activate coagulation factor XIII, which leads to covalent crosslinking of fibrin. The rate of proteolytic degradation of the fibrin polymer clot is decreased and mechanical stability is increased as a result of the covalent crosslinking of the polymer. Presence of fibronectin (U.S. Pat. No. 51,412 to Shane) and hyaluronic acid (U.S. Pat. No. 6,503,527 to whitmore) inhibit the proteolytic degradation, increase the fibril stability.
The fibrin polymer clot is porous with pore size range from 1 to 5 microns in mean diameter. It is too small to allow cell move freely. However, the micro-pore allows easy diffuse of nutrition, growth factor and hormone to maintain normal cellular activity and function. To move in the fibril matrices, cell has to secrete protease or stimulate the other cells such as macrophage to produce protease to degrade the fibril. Thus, fibrin polymer serves as a barrier for the stem cells till degradation of the fibrin polymer and/phagocytosis is complete.
Study using fibrin sealant as carrier for cell transplantation is a well known art. Several studies have used fibrin sealant to delivery chondrocytes to repair the chondrodefect in the knee. The resultant repair does not exhibit hypertrophy shown in cell transplant alone. The beauty of this art is that preparation is in liquid form after injection the fibrin polymerized in situ forming an ideal shape implant predetermined by the defect. Fibrin sealant also attempted to use as carrier for delivery of MSCs stem cell or BMPs to heal bony defect, interestingly, the bone growth in fibrin sealant group is not as robust as compare to other carrier such gelatin. However, fibrin sealant group does show more angiogenesis than other group (review articles). Fibrin sealant as carrier for angiogenesis growth factors exhibited potent agiogenesis potential in animal model and in vitro experiments (Andreas et al Molecular Medicine 2007 and US patent to Shane). Use of the fibrin sealants as carrier for stem cells use for osteogenesis is not fully explored ad is one aim of the present invention.
Bone screws are a basic part of modern internal fixation. They can be used independently, or, in particular types of implants, they can be used together with the implant. The common design of a screw consists of a tip, shaft, thread, and head. A round screw tip requires pretapping, whereas a fluted screw tip is self-tapping. The screw shaft is located between the head and the threaded portion of the screw. The screw thread is defined by its major or outside (thread diameter) and minor or root (shaft diameter) diameters, pitch, lead, and number of threads. The distance between adjacent threads is the pitch.
The 2 basic types of screws available for the variability of bone density are cortical and cancellous screws. Cortical screws are designed for compact diaphyseal bone, whereas cancellous screws are designed for the more trabecular metaphyseal bone. Cortical screws have a smaller major (thread) diameter, decreased pitch, and a shallower thread than cancellous screws. Cancellous screws typically have a larger major (thread) diameter and pitch and a greater difference between major and minor (shaft) diameters in comparison to cortical screws, providing more surface area for bone purchase. These screws are intended for use in metaphyseal fixation, where bone is softer.
A few new designs have made in screws to improve and expand their function in fracture fixation. Biodegradable screw is a very attractive design, since it eliminates possible hardware removal after fracture healing and potential to use it as target drug delivery. Extensive research has been focused in this area. Many drugs including BMPs and biodegradable materials have been tested and or proposed to form a drug delivery screws. Although the system has the capacity to carry significant amount of the drug, which able to sustain release for a long time, toxic reaction to the break down product and weak mechanic strength make the idea not feasible, except few condition where toxic reaction overweight the benefit of the treatment such as cancer (Brode, Integrascience).
Coating of a screw is another new development. In recent studies Mauni demonstrated that hydroxyl apetite (HA) coated screws improve fixation and outcomes in osteoporosis fracture (JOT 2009). Alex use Chitosan coated stainless steel screw to fix the contaminated fracture and demonstrated that gentamicin can be eluted from implant at detectable level for 96 hours. However, the drawback of this system is the dosage limitation during coating.
Porous material serve as a barrier to control the drug delivery is an ancient art for pharmaceutical industry. Author in 1990 have used hollowed polyporous hydroxyl apatite implant to control release of adriamycin, and observed a sustained release for over 45 days in vivo. Recent art of using porous material as out shell and degradable core to for drug delivery demonstrated zero order prolonged stable sustained release (Ca patent 2363902 to Fesrehaie). US Patent NO 2007161985 to Demakas et al disclosed a “porous screw” which has a channel inside had at least one pore on lateral wall, the pore is filled with PMMA and BMPs to allow bone structure to grow into the “porous screw”. However, BMP release from the PMMA is limited. US patent 20090192552 disclosed a special screw with expansible sleeve to anchor the bone while a hollow core used for injection of bone cement to enforce the fixation. The above idea is quite interesting. However, they are not suitable for delivery of stem cells, the other important component for bone formation and scope of clinical application is not clearly defined except spine surgery. Since bioactive agents release from above invention is limited and diffuse through full length of the screw, they may help bone in-growth to the implant at sub-cellular level to create biological fixation. Thus, this screw may be suitable for position screw fixation.
Since sustained high local concentration is critical for osteoinductive activity of BMPs and stem cell. A hollowed poly-porous screw with define chamber filling with biodegradable protein core is used in present invention as a barrier for targeted delivery and controlled release of BMP and stem cell to the injury site to facilitate tissue healing. Targeted delivery achieved to by using this new screw for fixation as lag screw, interference screw, and pedicle screw.